Two dedicated class C radical S-adenosylmethionine methyltransferases concertedly catalyse the synthesis of 7,8-dimethylmenaquinone.

Dimethylmenaquinone (DMMK), a prevalent menaquinone (MK) derivative of uncertain function, is characteristic for members of the class Coriobacteriia. Such bacteria are frequently present in intestinal microbiomes and comprise several pathogenic species. The coriobacterial model organism Adlercreutzia equolifaciens was used to investigate the enzymology of DMMK biosynthesis. A HemN-like class C radical S-adenosylmethionine methyltransferase (MenK2) from A. equolifaciens was produced in Wolinella succinogenes or Escherichia coli cells and found to methylate MK specifically at position C-7. In combination with a previously described MK methyltransferase (MqnK/MenK) dedicated to MK methylation at C-8, 7,8-DMMK6 was produced in W. succinogenes. The position of the two methyl groups was confirmed by two-dimensional NMR and midpoint redox potentials of 7-MMK6, 8-MMK6 and 7,8-DMMK6 were determined by cyclic voltammetry. A phylogenetic tree of MenK, MenK2 and HemN proteins revealed a Coriobacteriia-specific MenK2 clade. Using chimeric A. equolifaciens MenK/MenK2 proteins produced in E. coli it was shown that the combined linker and HemN domains determined the site-specificity of methylation. The results suggest that the use of MenK2 as a biomarker allows predicting the ability of DMMK synthesis in microbial species.

Minimal standards for describing new species belonging to the families Campylobacteraceae and Helicobacteraceae: Campylobacter, Arcobacter, Helicobacter and Wolinella spp.

Ongoing changes in taxonomic methods, and in the rapid development of the taxonomic structure of species assigned to the Epsilonproteobacteria have lead the International Committee of Systematic Bacteriology Subcommittee on the Taxonomy of Campylobacter and Related Bacteria to discuss significant updates to previous minimal standards for describing new species of Campylobacteraceae and Helicobacteraceae. This paper is the result of these discussions and proposes minimum requirements for the description of new species belonging to the families Campylobacteraceae and Helicobacteraceae, thus including species in Campylobacter, Arcobacter, Helicobacter, and Wolinella. The core underlying principle remains the use of appropriate phenotypic and genotypic methods to characterise strains sufficiently so as to effectively and unambiguously determine their taxonomic position in these families, and provide adequate means by which the new taxon can be distinguished from extant species and subspecies. This polyphasic taxonomic approach demands the use of appropriate reference data for comparison to ensure the novelty of proposed new taxa, and the recommended study of at least five strains to enable species diversity to be assessed. Methodological approaches for phenotypic and genotypic (including whole-genome sequence comparisons) characterisation are recommended.

Evaluation of the Helicobacteraceae in the oral cavity of dogs.

OBJECTIVE: To determine the Helicobacter spp present in the oral cavity of dogs and the relationship of those organisms with gastric Helicobacter spp to better define the potential for dog-human and dog-dog transmission. SAMPLE: Saliva and dental plaque from 28 dogs and gastric biopsy specimens from a subset of 8 dogs. PROCEDURES: PCR-based screening for Helicobacter spp was conducted on samples obtained from the oral cavity of 28 dogs. Comparative analysis was conducted on Helicobacteraceae 16S rDNA clone libraries from the oral cavity and stomach of a subset of 8 dogs (5 vomiting and 3 healthy) that had positive PCR results for Helicobacter spp. RESULTS: Helicobacteraceae DNA was identified in the oral cavity of 24 of 28 dogs. Analysis of cloned 16S rDNA amplicons from 8 dogs revealed that Wolinella spp was the most common (8/8 dogs) and abundant (52/57 [91%] clones) member of the Helicobacteraceae family in the oral cavity. Only 2 of 8 dogs harbored Helicobacter spp in the oral cavity, and 1 of those was coinfected with Helicobacter heilmannii and Helicobacter felis in samples obtained from the stomach and saliva. Evaluation of oral cavity DNA with Wolinella-specific PCR primers yielded positive results for 16 of 20 other dogs (24/28 samples were positive for Wolinella spp). CONCLUSIONS AND CLINICAL RELEVANCE: Wolinella spp rather than Helicobacter spp were the predominant Helicobacteraceae in the oral cavity of dogs. The oral cavity of dogs was apparently not a zoonotically important reservoir of Helicobacter spp that were non-Helicobacter pylori organisms.

Non-pylori Helicobacteraceae in the upper digestive tract of asymptomatic Venezuelan subjects: detection of Helicobacter cetorum-like and Candidatus Wolinella africanus-like DNA.

BACKGROUND: The spectrum of human non-pylori Helicobacter infections is expanding, with species such as H. heilmannii and H. felis occasionally being associated with gastritis. However, the existence of non-pylori Helicobacter colonization in asymptomatic subjects has not been evaluated. The aim of this study was to investigate whether Helicobacter species other than pylori are present in the upper digestive tract of asymptomatic human subjects. MATERIALS AND METHODS: A Helicobacteraceae-specific semi-nested polymerase chain reaction (PCR) assay was used to detect Helicobacter-like organisms in the upper digestive tract of 91 Venezuelan volunteers (aged 18-68 years, 41 females, 50 males). Species were identified by denaturing gradient gel electrophoresis analysis and sequencing of the PCR products. RESULTS: We detected DNA sharing 99-100% sequence identity in over 300-400 bp with the 16S rRNA genes of H. pylori, H. cetorum, and Candidatus Wolinella africanus in 76%, 16%, and 15% of the subjects, respectively. Multiple colonization was documented in 10% of the subjects: H. cetorum and Candidatus W. africanus (4%), H. pylori and Candidatus W. africanus (4%), and H. pylori and H. cetorum (2%). CONCLUSIONS: Our results suggest that non-pylori Helicobacteraceae colonization is relatively common in the Venezuelan asymptomatic population. This is the first report documenting the presence of H. cetorum DNA in the human upper digestive tract, and the second report of the recently discovered Candidatus W. africanus.

Multiplex PCR for the identification of Arcobacter and differentiation of Arcobacter butzleri from other arcobacters.

A multiplex polymerase chain reaction (PCR) assay to identify Arcobacter isolates and to distinguish A. butzleri from other arcobacters is described. The test uses two primer sets. Set I targets a section of the 16S rRNA genes of Arcobacter spp. Set II amplifies a portion of the 23S rRNA genes unique to A. butzleri. Specificity of the primer sets was evaluated using ATCC reference strains of A. butzleri, A. cryaerophilus, A. skirrowii, Bacteroides spp., Campylobacter spp., Helicobacter spp. and Wolinella succinogenes. Upon PCR amplification, all of the Arcobacter isolates yielded a 1223 bp product, whereas A. butzleri ATCC 49616 exhibited both a 1223 bp and a 686 bp product. No PCR product was observed for other closely related ATCC strains (n = 37). We next analyzed by multiplex PCR field strains of Arcobacter spp. (n = 108) which had been previously characterized to the species level by either DNA-DNA hybridization, dot blot hybridization, ribotyping or by serology. The 1223 bp multiplex PCR product identified all of the isolates as Arcobacter. The presence of both the 1223 and 686 bp amplicons identified 66 strains as A. butzleri. Speciation by multiplex PCR agreed with results obtained by the other methods. The multiplex PCR assay is specific, rapid and easy to interpret and, thus, will aid in elucidating the prevalence, epidemiology and zoonotic potential of Arcobacter.

Campylobacter showae sp. nov., isolated from the human oral cavity.

Nine Campylobacter-like strains were isolated from human gingival crevices and characterized. These strains were gram-negative, straight rods that were motile by means of multiple unipolar flagella. They were asaccharolytic and preferred an anaerobic atmosphere rather than a microaerophilic atmosphere for growth, and their growth was stimulated by formate and fumarate. These strains were biochemically similar to Campylobacter curvus and Campylobacter rectus, but were clearly distinguishable from these organisms by the number of flagella (two to five flagella at one end of the cell), by being catalase positive, by their whole-cell protein profiles, by their Western blot (immunoblot) patterns, and on the basis of DNA-DNA homology data. They could also be differentiated from the other species of the genus Campylobacter. The nine Campylobacter-like strains were compared with two strains (FDC 286 and VPI 10279) representing a previously described but unnamed Wolinella sp. The nine isolates and strains FDC 286 and VPI 10279 were found to be members of a single species. The 16S rRNA sequences of two strains of the newly identified species were compared with the rRNA sequences of 21 reference Campylobacter, Wolinella, and Helicobacter species in order to generate a phylogenetic tree. We propose the name Campylobacter showae for the newly identified strains; strain SU A4 (= ATCC 51146) is the type strain of this new species.

Review of chemosystematics: multivariate approaches to oral bacteria and yeasts.

There are several problems related to the classification and identification of bacterial and yeast species assigned to the genera Actinobacillus, Haemophilus, Pasteurella, Bacteroides, Prevotella, Porphyromonas, Campylobacter, Wolinella, Treponema, Candida, Torulopsis, and Saccharomyces, most of which belong to the resident oral microflora. The present review was written to demonstrate how multivariate analyses of data on cellular fatty acids, sugars, enzyme activities, and lysis kinetics during ethylenediaminetetraacetic acid (EDTA) and EDTA plus lysozyme treatment can be used to distinguish closely related species of these bacterial and yeast genera. With the exception of the Actinobacillus-Haemophilus-Pasteurella group, fatty acids were more discriminating than sugars. Enzymes from whole cells and outer membrane vesicles also contributed to taxonomic distinction. Apparently, chemosystematics, involving multivariate analyses, is a useful adjunct in oral microbial taxonomy.